U.S. patent application number 15/940152 was filed with the patent office on 2018-10-04 for coumarin-linked taxanes for detection and circumvention of cellular efflux.
The applicant listed for this patent is The University of Kansas. Invention is credited to Blake R. Peterson.
Application Number | 20180282314 15/940152 |
Document ID | / |
Family ID | 63671603 |
Filed Date | 2018-10-04 |
United States Patent
Application |
20180282314 |
Kind Code |
A1 |
Peterson; Blake R. |
October 4, 2018 |
COUMARIN-LINKED TAXANES FOR DETECTION AND CIRCUMVENTION OF CELLULAR
EFFLUX
Abstract
A compound can be a fluorescent taxane derivative having a
structure of Formula 1, salt, stereoisomer, tautomer, polymorph, or
solvate thereof. Formula 1 can be defined as: L, L-NH, or
L-NH--C.dbd.O is a linker; and R is a substituent, where --OH,
--O.sup.-, --NH.sub.2, and NH--CH.sub.3 are examples. Examples of
linkers can include glycine, beta-alanine, gamma-aminobutyric acid
(GABA). Pharmaceutical compositions can include the compound and a
pharmaceutically acceptable carrier, and may be configured for
intravenous injection. The fluorescent taxane derivative can be
used to treat cancer and non-cancer diseases. The fluorescent
taxane derivative can be used to monitor cellular efflux and
determine whether a cell will efflux paclitaxel. ##STR00001##
Inventors: |
Peterson; Blake R.;
(Lawrence, KS) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
The University of Kansas |
Lawrence |
KS |
US |
|
|
Family ID: |
63671603 |
Appl. No.: |
15/940152 |
Filed: |
March 29, 2018 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
62478954 |
Mar 30, 2017 |
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61P 35/00 20180101;
G01N 33/582 20130101; C07D 407/12 20130101 |
International
Class: |
C07D 407/12 20060101
C07D407/12; A61P 35/00 20060101 A61P035/00; G01N 33/58 20060101
G01N033/58 |
Goverment Interests
GOVERNMENT RIGHTS
[0002] This invention was made with government support under
GM103638 awarded by the National Institutes of Health. The
government has certain rights in the invention.
Claims
1. A compound comprising: a structure of Formula 9, salt,
stereoisomer, tautomer, polymorph, or solvate thereof; ##STR00017##
wherein: R3, R4, R5, R6, R7, and/or R8 are independently any
substituent so long as at least one of R3, R4, R5, R6, R7, and/or
R8 is a taxane coupled to a linker, wherein the linker is coupled
to Formula 9.
2. The compound of claim 1, comprising: a fluorescent taxane
derivative having a structure of Formula 1, salt, stereoisomer,
tautomer, polymorph, or solvate thereof; ##STR00018## wherein: L,
L-NH, or L-NH--C.dbd.O is the linker; R is selected from: (a)
--NH.sub.2, --NH-alkyl, --N(alkyl).sub.2, --NH.sub.2--R1,
--N(R1).sub.2 or --NR1R2, or combination thereof; (b) --C(O)R1a,
--C(O)CH(NR1bR1c)R1a, --C(O)CH(N(R1c)C(O)R1b)R1a,
--C(O)CH(N(R1c)C(O)OR1b)R1a, --C(O)CH(N(R1c)C(O)NR1bR1d)R1a,
--C(O)OR1a, --C(O)NR1bR1c, --C(NR1a)NR1bR1c, --P(O)(OR1a)R1d,
--CH2P(O)(OR1a)R1d, --S(O)R1a, --S(O)2R1a, --S(O)NR1bR1c, or
--S(O)2NR1bR1c; (c) C.sub.1-C.sub.24 alkyl, C.sub.2-C.sub.24
alkenyl, C.sub.2-C.sub.24 alkynyl, C.sub.3-C.sub.20 cycloalkyl,
C.sub.5-C.sub.20 aryl, C.sub.5-C.sub.20 polyaryl, C.sub.6-C.sub.24
alkaryl, C.sub.6-C.sub.24 aralkyl, heteroaryl, heterocyclyl,
hydrogen, halo, oxygen anion, hydroxy anion, hydroxyl, sulfhydryl,
C.sub.1-C.sub.24 alkoxy, C.sub.2-C.sub.24 alkenyloxy,
C.sub.2-C.sub.24 alkynyloxy, C.sub.5-C.sub.20 aryloxy, acyl,
C.sub.2-C.sub.24 alkylcarbonyl, C.sub.6-C.sub.20 arylcarbonyl,
acyloxy, C.sub.2-C.sub.24 alkoxycarbonyl, C.sub.6-C.sub.20
aryloxycarbonyl, halocarbonyl, C.sub.2-C.sub.24 alkylcarbonato,
C.sub.6-C.sub.20 arylcarbonato, carboxy, carboxylato, carbamoyl,
mono-(C.sub.1-C.sub.24 alkyl)-substituted carbamoyl,
di-(C.sub.1-C.sub.24 alkyl)-substituted carbamoyl, mono-substituted
arylcarbamoyl, thiocarbamoyl, carbamido, cyano, isocyano, cyanato,
isocyanato, isothiocyanato, azido, formyl, thioformyl, amino, mono-
and di-(C.sub.1-C.sub.24 alkyl)-substituted amino, mono- and
di-(C.sub.5-C.sub.20 aryl)-substituted amino, C.sub.2-C.sub.24
alkylamido, C.sub.6-C.sub.20 arylamido, imino, alkylimino,
arylimino, nitro, nitroso, sulfo, sulfonato, C.sub.1-C.sub.24
alkylsulfanyl, arylsulfanyl, C.sub.1-C.sub.24 alkylsulfinyl,
C.sub.5-C.sub.20 arylsulfinyl, C.sub.1-C.sub.24 alkylsulfonyl,
C.sub.5-C.sub.20 arylsulfonyl, phosphono, phosphonato, phosphinato,
phospho, or phosphino, or combinations thereof; and (d)
combinations thereof; wherein the R group is optionally substituted
by a substituent Q, which substituent Q is defined as R; and
wherein R1, R2, R1a, R1b, R1c, or R1d are each independently as
defined for R or independently hydrogen.
3. The compound of claim 2, wherein the linker is selected from:
C.sub.1-C.sub.24 alkyl, C.sub.2-C.sub.24 alkenyl, C.sub.2-C.sub.24
alkynyl, C.sub.3-C.sub.20 cycloalkyl, C.sub.5-C.sub.20 aryl,
C.sub.5-C.sub.20 polyaryl, C.sub.6-C.sub.24 alkaryl,
C.sub.6-C.sub.24 aralkyl, heteroaryl, heterocyclyl,
C.sub.1-C.sub.24 alkoxy, C.sub.2-C.sub.24 alkenyloxy,
C.sub.2-C.sub.24 alkynyloxy, C.sub.5-C.sub.20 aryloxy, acyl,
C.sub.2-C.sub.24 alkylcarbonyl, C.sub.6-C.sub.20 arylcarbonyl,
acyloxy, C.sub.2-C.sub.24 alkoxycarbonyl, C.sub.6-C.sub.20
aryloxycarbonyl, halocarbonyl, C.sub.2-C.sub.24 alkylcarbonato,
C.sub.6-C.sub.20 arylcarbonato, carboxy, carboxylato, carbamoyl,
mono-(C.sub.1-C.sub.24 alkyl)-substituted carbamoyl,
di-(C.sub.1-C.sub.24 alkyl)-substituted carbamoyl, mono-substituted
arylcarbamoyl, thiocarbamoyl, carbamido, amino, amino acid,
essential amino acid, L-amino acid, D-amino acid, non-essential
amino acid, glycine, beta-alanine, gamma-aminobutyric acid (GABA),
mono- and di-(C.sub.1-C.sub.24 alkyl)-substituted amino, mono- and
di-(C.sub.5-C.sub.20 aryl)-substituted amino, C.sub.2-C.sub.24
alkylamido, C.sub.6-C.sub.20 arylamido, or combinations thereof; or
##STR00019## wherein n is an integer.
4. The compound of claim 3, wherein the linker is selected from
glycine, beta-alanine, gamma-aminobutyric acid (GABA).
5. The compound of claim 3, wherein the linker is glycine.
6. The compound of claim 3, wherein: R is selected from --O.sup.-,
--OH, --NH.sub.2, --NH-alkyl, --N(alkyl).sub.2, --NH.sub.2--R1,
--N(R1).sub.2 or --NR1R2; R1 is C.sub.1-C.sub.24 alkyl; and R2 is
hydrogen or C.sub.1-C.sub.24 alkyl.
7. The compound of claim 6, wherein the R1 or alkyl is methyl and
R2 is hydrogen.
8. The compound of claim 6, wherein R is selected from --O.sup.-,
--OH, --NH-- methyl.
9. The compound of claim 6, wherein the linker is selected from
glycine, beta-alanine, gamma-aminobutyric acid (GABA).
10. The compound of claim 4, wherein the fluorescent taxane
derivative is selected from Formula 3, Formula 4, or Formula 5 or
salt, stereoisomer, tautomer, polymorph, or solvate thereof:
##STR00020##
11. The compound of claim 2, wherein the fluorescent taxane
derivative is selected from Formula 6, Formula 7, or Formula 8 or
salt, stereoisomer, tautomer, polymorph, or solvate thereof:
##STR00021## wherein n is an integer.
12. The compound of claim 2, wherein the taxane is selected from:
##STR00022##
13. The compound of claim 12, wherein the linker is selected from:
##STR00023## wherein n is an integer; and R is selected from
--O.sup.-, --OH, --NH.sub.2, --NH-alkyl, --N(alkyl).sub.2,
--NH.sub.2--R1, --N(R1).sub.2 or --NR1R2; R1 is C.sub.1-C.sub.24
alkyl; and R2 is hydrogen or C.sub.1-C.sub.24 alkyl.
14. The compound of claim 13, wherein: R is selected from
--O.sup.-, --OH, --NH-methyl; the linker is selected from glycine,
beta-alanine, gamma-aminobutyric acid (GABA).
15. A pharmaceutical composition comprising: the compound of claim
1; and a pharmaceutically acceptable carrier.
16. A method of treating cancer, comprising: administering the
compound of claim 1 to a subject having cancer.
17. The method of claim 16, wherein the cancer is Kaposi sarcoma,
cervical cancer, pancreatic cancer, ovarian cancer, breast cancer,
bladder cancer, prostate cancer, melanoma, esophageal cancer,
and/or lung cancer.
18. A method of studying of a microtubule, comprising: contacting
the compound of claim 1 to a microtubule in a cell; and monitoring
a functionality of the microtubule in the cell.
19. The method of claim 18, further comprising visualizing or
detecting the microtubule from fluorescence of the compound
attached to the microtubule.
20. A method of studying P-glycoprotein, comprising: contacting the
P-glycoprotein, cell having the P-glycoprotein, with the compound
of claim 1; and monitoring efflux or no efflux of one or more
substances from a cell having the P-glycoprotein.
21. The method of claim 20, further comprising visualizing or
detecting the efflux or no efflux of the compound from the cell
from fluorescence of the compound.
Description
CROSS-REFERENCE
[0001] This patent application claims priority to U.S. Provisional
Application No. 62/478,954 filed Mar. 30, 2017, which provisional
is incorporated herein by specific reference in its entirety.
BACKGROUND
[0003] Paclitaxel (Taxol.RTM.) is a natural product that can be
isolated from the bark of the yew tree Taxus brevifolia. Paclitaxel
has become an effective treatment for various types of cancers,
such as ovarian, breast, and lung. Taxol.RTM. has the biological
activity of binding the protein .beta.-tubulin and stabilizing
microtubules in the cytosol. Additionally, paclitaxel inhibits
microtubule polymerization dynamics and can trigger cellular death
via apoptosis. Although paclitaxel is widely used as a therapeutic,
some mechanisms underlying its selective cytotoxicity towards
cancer cells remain unknown. Paclitaxel and related compounds, such
as taxanes and taxoids, can exhibit antimitotic activity against
rapidly dividing cancer cell lines and xenografts that double every
1-12 days, suggesting that effects against rapidly proliferating
cells might provide selectivity against tumors in human patients.
However, these types of compounds also show major effects against
slow growing tumors (median doubling time of .about.147 days) in
patients, while sparing rapidly proliferating normal cells in bone
marrow, gut, and other tissues.
##STR00002##
[0004] Fluorescent analogues of paclitaxel have been used for
studying their anticancer effects. An example of such a compound
termed Flutax-2.RTM. includes paclitaxel linked at the 7-position
through a .beta.-Ala ester to the fluorophore Oregon Green (OG), as
shown below. The term Flutax-2.RTM. has also been used to describe
a related fluorescent probe where paclitaxel is linked to OG via an
L-Ala ester (termed here Flutax-2.RTM. (L-Ala)). The side-chain of
Taxol.RTM. has also been linked to the BODIPY fluorophore, but
these probes are generally not considered suitable for imaging of
living cells.
##STR00003##
[0005] At physiological pH, OG exists as a highly polar dianion. In
Flutax-2, this increases the polarity of paclitaxel by almost
1000-fold, which alters its calculated octanol-water
distribution/partition coefficient from c Log D.sub.pH7.4=2.9
(paclitaxel) to c Log D.sub.pH7.4=0.0 (Flutax-2.RTM.). Given that
most small molecule drugs are generally moderately hydrophobic (c
Log D.sub.pH7.4.about.2), which facilitates passive diffusion
across membranes, Flutax-2.RTM. differs substantially from
paclitaxel under physiological conditions.
[0006] Therefore, it may be advantageous to provide more
paclitaxel-like fluorescent probes.
SUMMARY
[0007] In one embodiment, the present invention includes taxanes
(e.g., paclitaxel is a taxane, and other taxoids are taxanes)
linked to a coumarin-derived fluorophore, such as Pacific Blue (PB)
or an analogue of PB. Additionally, the taxane-PB-fluorophores can
be used in methods similarly to paclitaxel or other fluorescent
taxanes. The taxanes may be linked to the coumarin-derived
fluorophore at any of carbons 3, 4, 5, 6, 7, or 8 as shown below,
and the other carbons may or may not include a substituent. In one
example, the taxane is linked to the coumarin-derived fluorophore
through carbon 3. In another example, the coumarin-derived
fluorophore may include substituents at carbons 6, 7, and 8. In
another example, the coumarin-derived fluorophore may include
halogens, such as fluorine at carbons 6 and 8. In another example,
the coumarin-derived fluorophore may include a substituent at
carbon 7, such as a hydroxyl, amine, alkyl-amine, or the like.
##STR00004##
BRIEF DESCRIPTION OF THE FIGURES
[0008] The foregoing and following information as well as other
features of this disclosure will become more fully apparent from
the following description and appended claims, taken in conjunction
with the accompanying drawings. Understanding that these drawings
depict only several embodiments in accordance with the disclosure
and are, therefore, not to be considered limiting of its scope, the
disclosure will be described with additional specificity and detail
through use of the accompanying drawings.
[0009] FIG. 1 shows the structures of paclitaxel, Flutax-2.RTM.,
and taxane PB fluorophore Compounds 1-3.
[0010] FIG. 1A shows reaction Scheme 1 to prepare the compounds of
FIG. 1.
[0011] FIG. 2 includes a graph that shows the quantification of the
affinities (Kd) of Compounds 1-3 for cross-linked microtubules by
enhancement of fluorescence, where binding studies were conducted
in aqueous GAB buffer (pH 6.5). PB was excited at 405 nm and
emitted photons were collected at and above 450 nm.
[0012] FIG. 3 shows a comparison of a published structure of
paclitaxel bound to .beta.-tubulin (PDB 3J6G, Panel A) with a model
(Panel B) of .beta.-tubulin docked to Compound 1. In Panel B, a
docking pose of Compound 1 is shown where the side chain
orientations are similar to those of paclitaxel in Panel A, and the
PB moiety of Compound 1 engages a neighboring pocket on the protein
surface.
[0013] FIG. 4A shows a graph of an analysis of cytotoxicity for the
compounds paclitaxel (Taxol.RTM.), Flutax-2.RTM., Compound 1
(PB-Gly-Taxol), Compound 2 (PB-.beta.-Ala-Taxol), and Compound 3
(PB-GABA-Taxol), where HeLa cells were treated with compounds in
the absence of the efflux inhibitor verapamil (25 .mu.M) for 48
hours (h) and cellular viability measured by flow cytometry.
[0014] FIG. 4B shows a graph of an analysis of cytotoxicity for the
compounds paclitaxel (Taxol.RTM.), Flutax-2.RTM., Compound 1
(PB-Gly-Taxol), Compound 2 (PB-.beta.-Ala-Taxol), and Compound 3
(PB-GABA-Taxol), where HeLa cells were treated with compounds in
the presence of the efflux inhibitor verapamil (25 .mu.M) for 48 h
and cellular viability measured by flow cytometry.
[0015] FIG. 5A includes an image from confocal laser scanning and
DIC microscopy of living HeLa cells treated with Compound 1 (1
.mu.M, 1 h) in the absence of verapamil, where the inset of FIG. 5A
shows the brightness was enhanced to reveal weak fluorescence of 1
resulting from efflux. Scale bar=25 microns.
[0016] FIG. 5B includes an image from confocal laser scanning and
DIC microscopy of living HeLa cells treated with Compound 1 (1
.mu.M, 1 h) in the presence of verapamil (100 .mu.M), where
inhibition of efflux by verapamil results in binding of Compound 1
to microtubules. Scale bar=25 microns.
[0017] FIG. 5C shows where cells were further treated with Compound
1 (1 .mu.M) verapamil (25 .mu.M) and Flutax-2.RTM. (5 .mu.M) for 1
hour to examine specificity. Arrows indicate unique punctate
fluorescence of Flutax-2.RTM. that indicates its lower specificity
for tubulin. Scale bar=25 microns.
[0018] FIG. 6A shows an image from confocal laser scanning and DIC
microscopy of living HCT-15 cells treated with the P-gp substrate
rhodamine 123 (1 .mu.M) with the vehicle only (left Fluor. and DIC
panels) and with the P-gp inhibitor verapamil (25 .mu.M, 1 h, right
Fluor. and DIC panels, resulting in enhanced cellular
fluorescence).
[0019] FIG. 6B shows an image from confocal laser scanning and DIC
microscopy of living HCT-15 cells treated with the P-gp substrate
Compound 1 (1 .mu.M) with the vehicle only (left Fluor. and DIC
panels), and with verapamil (25 .mu.M, 1 h, right Fluor. and DIC
panels, resulting in enhanced cellular fluorescence). Scale bar=25
microns.
[0020] FIG. 6C shows data from an analysis of living HCT-15 cells
treated with rhodamine 123 (1 .mu.M) (left panels) or Compound 1 (1
.mu.M) (right panels) by flow cytometry. Concentrations of added
verapamil are shown.
[0021] FIG. 6D shows data from an analysis of living HeLa cells
treated with rhodamine 123 (1 .mu.M) (left panels) or Compound 1 (1
.mu.M) (right panels) by flow cytometry. Concentrations of added
verapamil are shown.
[0022] FIG. 7 shows a graph of data for the uptake of Compound 1
into living HeLa, Ramos, Raji, HL-60, and Jurkat cell lines by flow
cytometry, illustrating increased uptake with addition of
increasing concentrations of verapamil.
[0023] FIG. 8A shows reaction Scheme 2 to prepare amino derivatives
of Pacific Blue that can be used to prepare other taxane PB
fluorophores.
[0024] FIG. 8B shows reaction Scheme 3 to prepare representative
taxane PB-amine (PB-NH.sub.2 and PB-NHMe) fluorophores.
[0025] FIG. 9 shows a graphical analysis of the toxicity of
paclitaxel (Taxol .RTM.), PB-Gly-Taxol (Compound 1),
PB-NHMe-Gly-Taxol (Compound 21), and PB-NH.sub.2-Gly-Taxol
(Compound 22).
[0026] FIG. 10A show a graph of absorbance for PB-NHMe-Gly-Taxol
and PB-NH.sub.2-Gly-Taxol in phosphate buffered saline (PBS, pH
7.4) and 1-octanol (as a mimic of cellular membranes).
[0027] FIG. 10B show a graph of emission for PB-NHMe-Gly-Taxol and
PB-NH.sub.2-Gly-Taxol in phosphate buffered saline (PBS, pH 7.4)
and 1-octanol (as a mimic of cellular membranes).
[0028] FIG. 11 shows images of a P-glycoprotein response to
PB-Gly-Taxol (1 Panels A and A1) and PB-NHMe-Gly-Taxol (Panels B
and B1 (10 nM), C and C1 (100 nM) and D and D1 (1 .mu.M) in living
HeLa cells. In panels A-D vehicle (DMSO, 0.1%) only was added as a
control. In panels A1-D1, verapamil (25 .mu.M) was added to inhibit
P-gp. Unlike PB-Gly-Taxol, PB-NHMe-Gly-Taxol and PB-NH2-Gly-Taxol
(data not shown) do not undergo efficient efflux by P-gp.
[0029] FIG. 12 shows PB-NHMe-Gly-Taxol (Compound 21) does not
visibly colocalize with Flutax-2.RTM. on microtubules in live HeLa
cells but rather extensively associates with internal membranes of
the endoplasmic reticulum. Differences in photophysical properties
of these fluorophores may prevent visualization of binding of
PB-NHMe-Gly-Taxol (Compound 21) to microtubules in living
cells.
[0030] FIG. 13 shows that PB-NHMe-Gly-Taxol (Compound 21)
specifically colocalizes with the fluorescent probe HRA (hexyl
resorufamine) in membranes of the endoplasmic reticulum of living
HeLa cells.
[0031] FIG. 14 shows confocal microscopy images of living HeLa
cells treated with PB-NHMe-Gly-Taxol (Compound 21, top panels) and
ER Tracker Blue-White DPX (bottom panels). Concentrations of probes
(left to right: 1 nM, 10 nM, 100 nM, 1 .mu.M). PB-NHMe-Gly-Taxol
shows greater potency in its accumulation in ER membranes.
DETAILED DESCRIPTION
[0032] In the following detailed description, reference is made to
the accompanying drawings, which form a part hereof. In the
drawings, similar symbols typically identify similar components,
unless context dictates otherwise. The illustrative embodiments
described in the detailed description, drawings, and claims are not
meant to be limiting. Other embodiments may be utilized, and other
changes may be made, without departing from the spirit or scope of
the subject matter presented herein. It will be readily understood
that the aspects of the present disclosure, as generally described
herein, and illustrated in the figures, can be arranged,
substituted, combined, separated, and designed in a wide variety of
different configurations, all of which are explicitly contemplated
herein.
[0033] Generally, the present technology includes taxanes (e.g.,
paclitaxel is a taxane, and other taxoids are derivatives of
taxanes) linked to a fluorophore derived from coumarin
(2H-chromene-2-one), such as Pacific Blue (PB) or derivative or
analogue of PB (PB derivative). For the purposes of this
disclosure, taxanes are intended to also cover taxoids. In this
application the abbreviation PB can refer to any coumarin-derived
structure. As such, the present disclosure provides taxane PB
fluorophores, referred to herein as TPBFs, which include any taxane
that is conjugated to a coumarin so as to retain the properties of
the taxane and the coumarin. In one aspect, the taxanes are
conjugated to a fluorescent coumarin or coumarin-derivative through
a linker, which are also referred to under the term TPBF.
[0034] An example of a taxane is paclitaxel, which can be
conjugated to the PB or PB derivative through a linker. The
resulting paclitaxel PB conjugate (e.g., including coumarins,
paclitaxel PB, and paclitaxel PB derivatives) can be substantially
smaller and less polar than Flutax-2.RTM. (c Log D.sub.pH7.4=2.0
for PB-Gly-Taxol, which is Compound 1 provided herein, see FIG. 1).
The other taxanes may be linked through any of the linkers to the
PB fluorophore to provide a compound that has the properties of the
taxane, such as paclitaxel properties, and the fluorescent
property.
[0035] In one embodiment, the TPBF compounds described herein can
be used to detect inhibitors of cellular efflux transporters and
other agents that affect these proteins. These transporters are of
interest for drug development projects. Existing substrates of
efflux transporters, such as P-glycoprotein (P-gp), lack
sensitivity. The TPBFs described herein are sensitive substrates of
this efflux transporter and bind microtubules in living cells. The
compounds have compatibility with flow cytometry, and other
fluorescent detection methods, and can be used to detect low levels
of P-gp activity undetectable with existing substrates. The TPBFs
have various uses such as those described herein, such as studying
the P-gp activity, among others.
[0036] Taxoids are a class of derivatives or analogues of
paclitaxel. The taxoids may have anticancer chemotherapeutic
properties as well as other properties of paclitaxel. As used
herein, taxoids may be synonymous with taxanes, and thereby the
taxanes recited herein include the taxoids. The taxoids class and
the taxanes class both include paclitaxel and docetaxel, among
others.
[0037] Pacific Blue is often provided as the Pacific Blue
succinimidyl ester, as shown below, for reaction with other
compounds. During coupling chemistry to couple the Pacific Blue to
a taxane, the procedure results in the Pacific Blue structure
provided below (Pacific Blue amide), which is then linked via the
nitrogen atom through a linker (e.g., L) to the taxane as shown. It
should be recognized that the oxygen anion of Pacific Blue may also
be present as a protonated phenol (--OH), and thereby the
structures of Pacific Blue are also intended to cover the
protonated form. For the purposes of clarity, the Pacific Blue
anion will be used to describe the presently technology; however,
the descriptions are also affirmatively indicated to also refer to
the structures including the protonated phenol species and other
protonated and deprotonated forms and salts.
##STR00005##
Pacific Blue succinimidyl ester
##STR00006##
Pacific Blue amide derivative shown as the anion (the deprotonated
form that predominates at physiological pH)
##STR00007##
Pacific Blue amide derivative as the phenol (protonated form that
may be isolated during synthesis)
##STR00008##
Taxane Pacific Blue anion
##STR00009##
Taxane Pacific Blue phenol
[0038] Now, it has been found that the Pacific Blue may be
derivatized, such as at the R group shown in Formula 1 below in
order to provide improved TPBFs. The R group may be any
substituent, such as reasonable substituents which are recited
herein. The R group may vary as described below. In one example,
the R group is hydroxyl (phenol). Another example is when the R
group includes a nitrogen, and thereby is a primary amine
(--NH.sub.2 or aniline) or alkyl secondary amine (--NH-alkyl), or
alkyl tertiary amine (--N(alkyl).sub.2). In another example, the R
group includes a nitrogen that is then linked to another
substituent, and thereby is a substituted secondary amine
(--NH.sub.2--R1), or di-substituted tertiary amine (--N(R1).sub.2
or --NR1R2), where the R1 group and/or R2 group is any substituent,
whether the same or different, such as shown in Formula 2.
##STR00010##
[0039] In one embodiment, the present technology includes a TPBF
compound, which is a fluorescent taxane derivative, having a
structure of Formula 1 or Formula 2 (shown above), salt,
stereoisomer, tautomer, polymorph, or solvate thereof. In one
aspect, the L, L-NH, or L-NH--C.dbd.O of Formula 1 or Formula 2 is
a linker. The R group can be any reasonable substituent.
[0040] In one embodiment, the R is selected from:
[0041] (a) --NH.sub.2, --NH-alkyl, --N(alkyl).sub.2,
--NH.sub.2--R1, --N(R1).sub.2 or --NR1R2, or combination
thereof;
[0042] (b) --C(O)R1a, --C(O)CH(NR1bR1c)R1a,
--C(O)CH(N(R1c)C(O)R1b)R1a, --C(O)CH(N(R1c)C(O)OR1b)R1a,
--C(O)CH(N(R1c)C(O)NR1bRld)R1a, --C(O)OR1a, --C(O)NR1bR1c,
--C(NR1a)NR1bR1c, --P(O)(OR1a)R1d, --CH2P(O)(OR1a)R1d, --S(O)R1a,
--S(O)2R1a, --S(O)NR1bR1c, or --S(O)2NR1bR1c;
[0043] (c) C.sub.1-C.sub.24 alkyl, C.sub.2-C.sub.24 alkenyl,
C.sub.2-C.sub.24 alkynyl, C.sub.3-C.sub.20 cycloalkyl,
C.sub.5-C.sub.20 aryl, C.sub.5-C.sub.20 polyaryl, C.sub.6-C.sub.24
alkaryl, C.sub.6-C.sub.24 aralkyl, heteroaryl, heterocyclyl,
hydrogen, halo, oxygen anion, hydroxyl anion, hydroxyl, sulfhydryl,
C.sub.1-C.sub.24 alkoxy, C.sub.2-C.sub.24 alkenyloxy,
C.sub.2-C.sub.24 alkynyloxy, C.sub.5-C.sub.20 aryloxy, acyl,
C.sub.2-C.sub.24 alkylcarbonyl, C.sub.6-C.sub.20 arylcarbonyl,
acyloxy, C.sub.2-C.sub.24 alkoxycarbonyl, C.sub.6-C.sub.20
aryloxycarbonyl, halocarbonyl, C.sub.2-C.sub.24 alkylcarbonato,
C.sub.6-C.sub.20 aryl carbonato, carboxy, carboxylato, carbamoyl,
mono-(C.sub.1-C.sub.24 alkyl)-substituted carbamoyl,
di-(C.sub.1-C.sub.24 alkyl)-substituted carbamoyl, mono-substituted
arylcarbamoyl, thiocarbamoyl, carbamido, cyano, isocyano, cyanato,
isocyanato, isothiocyanato, azido, formyl, thioformyl, amino, mono-
and di-(C.sub.1-C.sub.24 alkyl)-substituted amino, mono- and
di-(C.sub.5-C.sub.20 aryl)-substituted amino, C.sub.2-C.sub.24
alkylamido, C.sub.6-C.sub.20 arylamido, imino, alkylimino,
arylimino, nitro, nitroso, sulfo, sulfonato, C.sub.1-C.sub.24
alkylsulfanyl, arylsulfanyl, C.sub.1-C.sub.24 alkylsulfinyl,
C.sub.5-C.sub.20 arylsulfinyl, C.sub.1-C.sub.24 alkylsulfonyl,
C.sub.5-C.sub.20 arylsulfonyl, phosphono, phosphonato, phosphinato,
phospho, or phosphino, or combinations thereof; as well as
combinations of (a), (b), and (c).
[0044] In one embodiment, under Formula 1 or Formula 2, the R group
is optionally substituted by a substituent Q, which substituent Q
is defined as R.
[0045] In one embodiment, R1, R2, R1a, R1b, R1c, or R1d are each
independently as defined for R or independently hydrogen. In one
aspect, R is not hydrogen. In one aspect, one or both of R1 and R2
can be hydrogen. In one aspect, one of R1 and R2 can be hydrogen,
and the other an alkyl, such as methyl, ethyl, propyl, butyl,
pentyl or the like, whether straight or branched or cyclic.
[0046] In one embodiment, the linker is selected from:
C.sub.1-C.sub.24 alkyl, C.sub.2-C.sub.24 alkenyl, C.sub.2-C.sub.24
alkynyl, C.sub.3-C.sub.20 cycloalkyl, C.sub.5-C.sub.20 aryl,
C.sub.5-C.sub.20 polyaryl, C.sub.6-C.sub.24 alkaryl,
C.sub.6-C.sub.24 aralkyl, heteroaryl, heterocyclyl,
C.sub.1-C.sub.24 alkoxy, C.sub.2-C.sub.24 alkenyloxy,
C.sub.2-C.sub.24 alkynyloxy, C.sub.5-C.sub.20 aryloxy, acyl,
C.sub.2-C.sub.24 alkylcarbonyl, C.sub.6-C.sub.20 arylcarbonyl,
acyloxy, C.sub.2-C.sub.24 alkoxycarbonyl, C.sub.6-C.sub.20
aryloxycarbonyl, halocarbonyl, C.sub.2-C.sub.24 alkylcarbonato,
C.sub.6-C.sub.20 aryl carb onato, carboxy, carboxylato, carb amoyl,
mono-(C.sub.1-C.sub.24 alkyl)-substituted carbamoyl,
di-(C.sub.1-C.sub.24 alkyl)-substituted carbamoyl, mono-substituted
arylcarbamoyl, thiocarbamoyl, carbamido, amino, amino acid,
essential amino acid, non-essential amino acid, L-amino acid,
D-amino acid, glycine, beta-alanine, gamma-aminobutyric acid
(GABA), mono- and di-(C.sub.1-C.sub.24 alkyl)-substituted amino,
mono- and di-(C.sub.5-C.sub.20 aryl)-substituted amino,
C.sub.2-C.sub.24 alkylamido, C.sub.6-C.sub.20 arylamido, or
combinations thereof.
[0047] In one aspect, the linker may be one of linkers L1, L2, L3,
or L4 as shown below, wherein n is an integer. It should be
recognized that the linker extends between the taxane and the
fluorescent PB fluorophore, and thereby the determination of the
end of the taxane and beginning of the linker as well as the end of
the linker and beginning of the PB fluorophore may be defined.
Accordingly, linkers L1, L2, L3, or L4 account for the different
possibilities of the end of the taxane and beginning of the linker
as well as the end of the linker and beginning of the PB
fluorophore. Preferably, the linker is L4 so that the taxane is
considered to include the terminal oxygen that links to the linker,
and the PB fluorophore is considered to include the carboxyl
(C.dbd.O). For the linker, n may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,
11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24. The
linker may be attached to carbon atoms 3, 4, 5, 6, 7, or 8 of the
coumarin.
##STR00011##
[0048] In one embodiment, the linker is selected from glycine,
beta-alanine, gamma-aminobutyric acid (GABA). In one example, the
linker is glycine.
[0049] In one embodiment, R is selected from --O.sup.-, --OH,
--NH.sub.2, --NH-alkyl, --N(alkyl).sub.2, --NH.sub.2--R1,
--N(R1).sub.2 or --NR1R2; R1 is C.sub.1-C.sub.24 alkyl; and R2 is
hydrogen or C.sub.1-C.sub.24 alkyl. In one aspect, the R1 or alkyl
is methyl and R2 is hydrogen. In one aspect, R is selected from
--O.sup.-, --OH, --NH-methyl. In one aspect of this embodiment for
R, the linker is selected from glycine, beta-alanine,
gamma-aminobutyric acid (GABA).
[0050] In one embodiment, the fluorescent taxane derivative is
selected from Formula 3, Formula 4, or Formula 5 or salt,
stereoisomer, tautomer, polymorph, or solvate thereof. The linker
can be any linker described herein. Also, the taxane can be any
taxane, such as those described herein or otherwise known.
##STR00012##
[0051] In one embodiment, the fluorescent taxane derivative is
selected from Formula 6, Formula 7, or Formula 8 or salt,
stereoisomer, tautomer, polymorph, or solvate thereof. The "n" of
the linker can be any integer described herein. Also, the taxane
can be any taxoid, such as those described herein or otherwise
known.
##STR00013##
[0052] While the taxane can be any known or developed taxane, such
as those described herein, certain taxanes are provided herein as
examples, including Paclitaxel, Baccatin III, 10-Deacetylbaccatin
III, and Docetaxel, as shown below. The point of conjugation of the
taxanes are shown.
##STR00014##
[0053] In one embodiment, the linker is one of L1, L2, L3, or L4
and R is selected from --O.sup.- (oxygen anion), --OH, --NH.sub.2,
--NH-alkyl, --N(alkyl).sub.2, --NH.sub.2-R1, --N(R1).sub.2 or
--NR1R2; R1 is C.sub.1-C.sub.24 alkyl; and R2 is hydrogen or
C.sub.1-C.sub.24 alkyl.
[0054] In one embodiment, R is selected from --O.sup.-, --OH,
--NH-methyl; and the linker is selected from glycine, beta-alanine,
gamma-aminobutyric acid (GABA). In one embodiment, it is preferred
that the linker is glycine.
[0055] Optionally, the linker between the taxane or taxoid and the
pacific blue can be substituted with a substituent. Optionally,
either fluorine of the pacific blue or the OH of the pacific blue
or any other hydrogen of the coumarin core structure can be
substituted with different substituents. These substituents can
independently include hydrogen, alkyl, alkenyl, alkynyl, aryl,
alkaryl, aralkyl, halo, hydroxyl, sulfhydryl, alkoxy, alkenyloxy,
alkynyloxy, aryloxy, acyl, alkylcarbonyl, aryl carbonyl, acyloxy,
alkoxycarbonyl, aryloxycarbonyl, halocarbonyl, alkylcarbonato,
arylcarbonato, carboxy, carboxylato, carbamoyl,
mono-(alkyl)-substituted carbamoyl, di-(alkyl)-substituted
carbamoyl, mono-substituted arylcarbamoyl, thiocarbamoyl,
carbamido, cyano, isocyano, cyanato, isocyanato, isothiocyanato,
azido, formyl, thioformyl, amino, mono- and di-(alkyl)-substituted
amino, mono- and di-(aryl)-substituted amino, alkylamido arylamido,
imino, alkylimino, arylimino, nitro, nitroso, sulfo, sulfonato,
alkylsulfanyl, arylsulfanyl, alkylsulfinyl, arylsulfinyl,
alkylsulfonyl, aryl sulfonyl, phosphono, phosphonato, phosphinato,
phospho, phosphino, any with or without hetero atoms, derivatives
thereof, and combinations thereof. Optionally, the linker between
the taxane or taxoid and the pacific blue substance can include
straight aliphatics, branched aliphatics, cyclic aliphatics,
substituted aliphatics, unsubstituted aliphatics, saturated
aliphatics, unsaturated aliphatics, aromatics, polyaromatics,
substituted aromatics, hetero-aromatics, amines, primary amines,
secondary amines, tertiary amines, aliphatic amines, carbonyls,
carboxyls, carbamates, amides, esters, amino acids, peptides,
polypeptides, derivatives thereof, substituted or unsubstituted, or
combinations thereof as well as other well-known chemical
substituents.
[0056] In another option, the linker between the taxane or taxoid
and the coumarin can be substituted with a substituent, and/or
either fluorine (F) or other hydrogen (H) atom or the hydroxyl (OH)
of the coumarin core structure of the Pacific Blue coumarin
derivative shown can be substituted with different substituents.
These substituents can be independently any one or more of the
substituents selected from the group of hydrogen, C.sub.1-C.sub.24
alkyl, C.sub.2-C.sub.24 alkenyl, C.sub.2-C.sub.24 alkynyl,
C.sub.6-C.sub.20 aryl, C.sub.7-C.sub.24 alkaryl, C.sub.7-C.sub.24
aralkyl, halo, hydroxyl, sulfhydryl, C.sub.1-C.sub.24 alkoxy,
C.sub.2-C.sub.24 alkenyloxy, C.sub.2-C.sub.24 alkynyloxy,
C.sub.6-C.sub.20 aryloxy, acyl (including C.sub.2-C.sub.24
alkylcarbonyl (--CO-alkyl) and C.sub.7-C.sub.20 arylcarbonyl
(--CO-aryl)), acyloxy (--O-acyl), C.sub.2-C.sub.24 alkoxycarbonyl
(--(CO)--O-alkyl), C.sub.7-C.sub.20 aryloxycarbonyl
(--(CO)--O-aryl), halocarbonyl (--CO)--X where X is halo),
C.sub.2-C.sub.24 alkylcarbonato (--O--(CO)--O-alkyl),
C.sub.7-C.sub.20 arylcarbonato (--O--(CO)--O-aryl), carboxy
(--COOH), carboxylato (--COO.sup.-), carbamoyl (--(CO)--NH.sub.2),
mono-(C.sub.1-C.sub.24 alkyl)-substituted carbamoyl
(--(CO)--NH(C.sub.1-C.sub.24 alkyl)), di-(C.sub.1-C.sub.24
alkyl)-substituted carbamoyl (--(CO)--N(C.sub.1-C.sub.24
alkyl).sub.2), mono-substituted arylcarbamoyl (--(CO)--NH-aryl),
di-substituted arylcarbamoyl (--(CO)--NH-aryl)2, thiocarbamoyl
(--(CS)--NH.sub.2), mono-(C.sub.1-C.sub.24 alkyl)-substituted
thiocarbamoyl (--(CS)--NH(C.sub.1-C.sub.24 alkyl)),
di-(C.sub.1-C.sub.24 alkyl)-substituted thiocarbamoyl
(--(CS)--N(C.sub.1-C.sub.24 alkyl).sub.2), mono-substituted
arylthiocarbamoyl (--(CS)--NH-aryl), di-substituted
arylthiocarbamoyl (--(CS)--NH-aryl).sub.2, carbamido
(--NH--(CO)--NH.sub.2),), mono-(C.sub.1-C.sub.24 alkyl)-substituted
carbamido (--NH--(CO)--NH(C.sub.1-C.sub.24 alkyl)),
di-(C.sub.1-C.sub.24 alkyl)-substituted carbamido
(--NH--(CO)--N(C.sub.1-C.sub.24 alkyl).sub.2), mono-substituted
aryl carbamido (--NH--(CO)--NH-aryl), di-substituted aryl carbamido
(--NH--(CO)--N-(aryl).sub.2) cyano(--C.ident.N), isocyano
(--N.sup.+.ident.C.sup.-), cyanato (--O--C.ident.N), isocyanato
(--O--N.sup.+.ident.C.sup.-), thiocyanato (--S--C.ident.N),
isothiocyanato (--S--N.sup.-.ident.C.sup.-), azido
(--N.dbd.N.sup.+.dbd.N.sup.-), formyl (--(CO)--H), thioformyl
(--(CS)--H), amino (--NH.sub.2), mono- and di-(C.sub.1-C.sub.24
alkyl)-substituted amino, mono- and di-(C.sub.6-C.sub.20
aryl)-substituted amino, C.sub.2-C.sub.24 alkylamido
(--NH--(CO)-alkyl), C.sub.6-C.sub.20 arylamido (--NH--(CO)-aryl),
imino (--CR.dbd.NH where R is hydrogen, C.sub.1-C.sub.24 alkyl,
C.sub.6-C.sub.20 aryl, C.sub.7-C.sub.24 alkaryl, C.sub.7-C.sub.24
aralkyl, etc.), alkylimino (--CR.dbd.N(alkyl), where R=hydrogen,
C.sub.1-C.sub.24 alkyl, aryl, alkaryl, aralkyl, etc.), arylimino
(--CR.dbd.N(aryl), where R=hydrogen, alkyl, aryl, alkaryl, etc.),
nitro (--NO.sub.2), nitroso (--NO), sulfonic acid (--SO.sub.2--OH),
sulfonato (--SO.sub.2--O.sup.-)' C.sub.1-C.sub.24 alkylsulfanyl
(--S-alkyl; also termed "alkylthio"), C.sub.6-C.sub.20 arylsulfanyl
(--S-aryl; also termed "arylthio"), C.sub.1-C.sub.24 alkylsulfinyl
(--(SO)-alkyl), C.sub.6-C.sub.20 arylsulfinyl (--(SO)-aryl),
C.sub.1-C.sub.24 alkylsulfonyl (--SO.sub.2-alkyl), C.sub.6-C.sub.20
arylsulfonyl (--SO.sub.2-aryl), phosphono (--P(O)(OH).sub.2),
phosphonato (--P(O)(O.sup.-).sub.2), phosphinato (--P(O)(O--)),
phospho (--PO.sub.2), phosphino (--PH.sub.2), any with or without
hetero atoms (e.g., N, O, P, S, or other) where the hetero atoms
can be substituted (e.g., hetero atom substituted for carbon in
chain or ring) for the carbons or in addition thereto (e.g., hetero
atom added to carbon chain or ring) swapped, derivatives thereof,
and combinations thereof.
[0057] Additionally, the coumarin-derived fluorophore may be
substituted at one or more of the carbon atoms 3, 4, 5, 6, 7, or 8
of the coumarin, which may include the taxane being attached to one
of carbon atoms 3, 4, 5, 6, 7, or 8 of the coumarin, and no
substituents or one or more substituents being attached to one or
more of the other carbon atoms of carbon atoms 3, 4, 5, 6, 7, or 8
of the coumarin. Accordingly, the present technology includes
fluorescent coumarin derivative, having a structure of Formula 9,
salt, stereoisomer, tautomer, polymorph, or solvate thereof. The
R3, R4, R5, R6, R7, and/or R8 groups can be any reasonable
substituent so long as one of R3, R4, R5, R6, R7, and/or R8 is a
taxane and linker (e.g., L) linked thereto (e.g., taxane-L-). The
taxane can be any taxane, such as the taxanes shown herein. The
linker can be any linker, such as the linker being one of linkers
L1, L2, L3, or L4 as shown here or a linker as defined herein. The
R3, R4, R5, R6, R7, and/or R8 groups can be as defined for R, R2,
and R2, and may be optionally substituted by a substituent Q, which
substituent Q is the same as defined as R.
##STR00015##
[0058] In one embodiment, the present technology includes a
pharmaceutical composition having: the compound of one of the
embodiments; and a pharmaceutically acceptable carrier. The
pharmaceutically acceptable carrier can be any known carrier, such
as those described herein. The pharmaceutical composition can be
used in any of the methods described herein or for any use of a
taxane, such as paclitaxel. Additionally, the use as a
pharmaceutical composition allows for imaging and visualization of
the localization of the taxane by the fluorescence of the PB
fluorophore.
[0059] In one embodiment, the taxane PB fluorophore can be used in
a method of treating cancer. Such a method can include
administering the compound of one of the embodiments to a subject
having cancer. For example, the cancer is Kaposi sarcoma, cervical
cancer, pancreatic cancer, ovarian, breast, and/or lung cancer;
however, the compound may be useful for other cancers. Often, the
administration of the pharmaceutical composition is intravenous
injection; however, other modes of administration such as oral
administration are known or may be developed. In one embodiment,
due to a possible allergic reaction, the compound can be
co-administered with an anti-allergy medicine.
[0060] Beneficially, the fluorescent nature of the taxane PB
fluorophore may be used for determining the effectiveness of the
treatment by obtaining biological samples, such as via a biopsy of
the cancer, including a tumor and/or surrounding tissue to
determine whether the distribution within the biological sample.
Samples that have higher amounts in the tumors may be more
effective. Also, the protocol of visualizing the fluorophore can be
used in any method of administering the compound to a subject, and
obtaining biological samples from various locations, organs,
tissues, or anatomical body parts. This may also be used to monitor
tumor metastasis. As such, the translocalization of cancerous cells
may be monitored, such as visualized, by monitoring the movement of
cancer cells in a body or identifying different location of the
cancer cells within the body (e.g., via biopsy or fluorescent
imaging catheter).
[0061] The biological activity of paclitaxel arrests cell cycle and
induces cell death by stabilizing microtubules and interfering with
microtubule disassembly in cell division. Recently, it has been
found that low-dose paclitaxel may be used in treating non-cancer
diseases, such as skin disorders, renal and hepatic fibrosis,
inflammation, axon regeneration, limb salvage, and coronary artery
restenosis. Thus, the present technology may be used in method for
treating one or more of these non-cancer diseases.
[0062] In one embodiment, a method of studying microtubules can
include: contacting the compound of one of the embodiments with
microtubules within a cell or extracted from cells; and/or
monitoring the functionality of microtubules in cells. In one
aspect, the method can include visualizing microtubules from
fluorescence of the compound bound to microtubules.
[0063] In one embodiment, a method of studying a P-glycoprotein can
include: studying interactions with purified P-glycoprotein, or in
cell(s) containing P-glycoprotein, with the compound of one of the
embodiments; and monitoring efflux or no efflux of one or more
substances from a cell having the P-glycoprotein. In one aspect,
the method can include visualizing the efflux or no efflux of the
compound from the cell from changes in fluorescence of the
compound. In one aspect, this protocol can be used to determine
whether or not a cell, such as a cancerous cell, can efflux the
taxane PB fluorophore. If the cell cannot efflux the taxane PB
fluorophore, then paclitaxel may be more suitable for treatment of
the cancerous cell. If the cell can efflux the taxane PB
fluorophore, then paclitaxel may not be as suitable for treatment
of the cancerous cells. Such monitoring can be done with a cell
culture of a biopsy, in vitro, ex vivo, or in vivo. Taxane PB
compounds may be used for high-throughput screening or high-content
screening or other screening assays for the discovery of inhibitors
or modulators of P-glycoprotein or other efflux transporters that
use them as substrates.
[0064] Whereas monitoring the efflux of the taxane PB fluorophore
may be used for cancerous cells, it may also be used with any cells
that are involved with a non-cancerous disease state of skin
disorders, renal and hepatic fibrosis, inflammation, axon
regeneration, limb salvage, and coronary artery restenosis. In one
aspect, this protocol can be used to determine whether or not a
cell can efflux the taxane PB fluorophore. If the cell cannot
efflux the taxane PB fluorophore, then paclitaxel may be suitable
to treat the cell. If the cell can efflux the taxane PB
fluorophore, then paclitaxel may not be suitable to treat the
cells. Such monitoring can be done with a cell culture of a biopsy,
in vitro, ex vivo, or in vivo.
[0065] These compounds can be prepared as pharmaceutical
compositions. Pharmaceutical compositions include, without
limitation, lyophilized powders or aqueous or non-aqueous sterile
injectable solutions or suspensions, which may further contain
antioxidants, buffers, bacteriostats and solutes that render the
compositions substantially compatible with the tissues or the blood
of an intended recipient. Other components that may be present in
such compositions include water, surfactants (e.g., Tween.RTM. and
Cremophor EL), alcohols, polyols, glycerin and vegetable oils, for
example. Extemporaneous injection solutions and suspensions may be
prepared from sterile powders, granules, tablets, or concentrated
solutions or suspensions. The composition may be supplied, for
example but not by way of limitation, as a lyophilized powder which
is reconstituted with sterile water or saline prior to
administration to the patient.
[0066] Suitable pharmaceutically acceptable carriers include
essentially chemically inert and nontoxic compositions that do not
interfere with the effectiveness of the biological activity of the
pharmaceutical composition. Examples of suitable pharmaceutical
carriers include, but are not limited to, water, saline solutions,
glycerol solutions, ethanol, N-(1
(2,3-dioleyloxy)propyl)N,N,N-trimethylammonium chloride (DOTMA),
diolesyl-phosphotidyl-ethanolamine (DOPE), and liposomes. Such
compositions should contain a therapeutically effective amount of
the compound, together with a suitable amount of carrier so as to
provide the form for direct administration to the patient.
[0067] The compositions described herein can be administered for
example, by parenteral, intravenous, subcutaneous, intramuscular,
intracranial, intraorbital, ophthalmic, intraventricular,
intracapsular, intraspinal, intracisternal, intraperitoneal,
intranasal, aerosol or oral administration. Common carriers or
excipients can be used for preparing pharmaceutical compositions
designed for such routes of administration.
[0068] Experimental
[0069] FIG. 1 shows the structures of paclitaxel, Flutax-2.RTM.,
and taxane PB fluorophores Compounds 1-3.
[0070] The compounds described herein may be synthesized via the
protocol in Scheme 1, which protocol may be modified depending on
the desired taxane PB fluorophore to be prepared. Scheme 1 is shown
in FIG. 1A. The compounds are identified by the compound numbers.
Since taxoids modified at the 7-position can retain high affinity
for microtubules, PB was linked at this position by amino acids
that differ subtly in the number of methylenes between the amine
and the carbonyl. Optical spectroscopy confirmed that Compounds 1-3
are similar to PB, whereas Flutax-2.RTM. is similar to OG. It
should be noted that the reaction in Scheme 1 is shown to produce
the Compounds 1-3 having the R group under Formula 1 being the
hydroxyl; however, it should be recognized that the hydrogen of the
hydroxyl may be deprotonated so as to be the structures shown in
FIG. 1. Accordingly, the same Compounds 1-3 may have the R group as
the hydroxyl or oxygen anion. FIG. 1A shows the reaction Scheme 1
that is performed to prepare the taxane (e.g., paclitaxel) into the
taxane PB fluorophores under Formula 1. Also, it should be
recognized that any taxane may be prepared as shown to be the
reagent in order to prepare a wide variety of taxane PB
fluorophores.
[0071] Experiments were conducted to obtain the measured affinities
of Compounds 1-3 for cross-linked microtubules from bovine brain
using a fluorescence enhancement method (FIG. 2). FIG. 2 includes a
graph that shows the quantification of the affinities (Kd) of
Compounds 1-3 for cross-linked microtubules by enhancement of
fluorescence, where binding studies were conducted in aqueous GAB
buffer (pH 6.5). PB was excited at 405 nm and emitted photons were
collected at and above 450 nm. Curve fitting of equilibrium binding
curves yielded apparent Kd values of 34.+-.6 nM for PB-Gly-Taxol
(Compound 1), 63.+-.8 nM for PB-.beta.-Ala-Taxol (Compound 2), and
265.+-.54 nM for PB-GABA-Taxol (Compound 3) in GAB buffer. Addition
of excess paclitaxel (10 .mu.M) blocked this fluorescence
enhancement, demonstrating that Compounds 1-3 bind .beta.-tubulin
at the same site as paclitaxel (data not shown).
[0072] The differences in the affinities of Compounds 1-3 were
studied using Autodock vina to the paclitaxel-binding site from a
recent Cryo-EM structure of .beta.-tubulin bound to paclitaxel.
These modeling studies suggested that linked fluorophores may
favorably insert into a pocket near the paclitaxel-binding site
(FIG. 3). As a result, using shorter linkers, such as n being 1 or
glycine, may be advantageous. FIG. 3 shows a comparison of a
published structure of paclitaxel bound to .beta.-tubulin (PDB
3J6G, Panel A) with a model (Panel B) of .beta.-tubulin docked to
Compound 1. In Panel B, a docking pose of Compound 1 is shown where
the side chain orientations are similar to those of paclitaxel in
Panel A, and the PB moiety of Compound 1 engages a neighboring
pocket on the protein surface.
[0073] The cytotoxicity of the taxane PB fluorophores towards HeLa
cervical carcinoma cells (FIGS. 4A and 4B) was evaluated. These
cell lines were treated for 48 h, cellular viability was analyzed
by flow cytometry, and paclitaxel was used as a positive control.
HCT-15 cells overexpress P-glycoprotein (P-gp, MDR1, ABCB1), and
because paclitaxel is a substrate of this efflux transporter, the
studies further measured cytotoxicity in the presence of the P-gp
inhibitor verapamil (FIG. 4B). FIG. 4A shows a graph of an analysis
of cytotoxicity for the compounds paclitaxel (Taxol.RTM.),
Flutax-2.RTM., Compound 1 (PB-Gly-Taxol), Compound 2
(PB-.beta.-Taxol), and Compound 3 (PB-GABA-Taxol). HeLa cells were
treated with compounds in the absence of verapamil (25 .mu.M) for
48 h and cellular viability measured by flow cytometry. FIG. 4B
shows a graph of an analysis of cytotoxicity for the compounds
paclitaxel (Taxol.RTM.), Flutax-2.RTM., Compound 1 (PB-Gly-Taxol),
Compound 2 (PB-.beta.-Taxol), and Compound 3 (PB-GABA-Taxol). HeLa
cells were treated with compounds in the presence of verapamil (25
.mu.M) for 48 h and cellular viability measured by flow
cytometry.
[0074] Among the fluorescent probes, Compound 1 was uniquely toxic,
with IC50=120 nM in the absence of verapamil, and IC50=60 nM in the
presence of verapamil (25 .mu.M) in HeLa cells. In HCT-15 cells,
verapamil (25 .mu.M) enhanced the toxicity of Compound 1 by 41-fold
(from IC50=3.7 .mu.M to IC50=90 nM). Control experiments confirmed
that verapamil itself (IC50>75 .mu.M at 48 h) did not contribute
to these cytotoxic effects. HeLa cells express low levels of P-gp
compared to HCT-15 cells, and this enhanced cytotoxicity mediated
by verapamil indicates that Compounds 1-3 are highly efficient
substrates of this drug efflux transporter. Flutax-2.RTM. is known
to be a substrate of P-gp, but in HeLa cells treated with
verapamil, Flutax-2.RTM. was the least cytotoxic (IC50=1310 nM),
likely due to its higher polarity and associated lower cellular
permeability or off-target effects.
[0075] To examine the subcellular distribution of Compound 1, HeLa
cells were imaged by confocal laser scanning microscopy (FIGS. 5A,
5B, and 5C). After treatment of cells with 1 (1 .mu.M, 1 h),
cotreatment with verapamil dramatically enhanced cellular
fluorescence in a dose dependent manner (FIGS. 5A, 5B, and 5C),
revealing intricate networks of microtubules in living cells. FIG.
5A includes an image from confocal laser scanning and DIC
microscopy of HeLa cells treated with Compound 1 (1 .mu.M, 1 h) in
the absence of verapamil (100 .mu.M), where the inset of FIG. 5A
shows the brightness was enhanced to reveal the weak fluorescence
of Compound 1 resulting from efflux. FIG. 5B includes an image from
confocal laser scanning and DIC microscopy of HeLa cells treated
with Compound 1 (1 .mu.M, 1 h) in the presence of verapamil (100
.mu.M). FIG. 5C shows where cells were further treated with
Compound 1 (1 .mu.M) verapamil (25 .mu.M) and Flutax-2.RTM. (5
.mu.M) for 1 hour to examine specificity. Arrows indicate punctate
fluorescence of Flutax-2.RTM. resulting from off-target effects.
The distinct spectral profiles of PB and OG were used to further
examine colocalization of Compound 1 and Flutax-2.RTM. (FIG. 5C).
Whereas Compound 1 bound microtubules with very high specificity,
Flutax-2.RTM. additionally conferred punctate fluorescence that did
not co-localize with Compound 1 on microtubules.
[0076] Overexpression of P-gp frequently confers resistance to the
antiproliferative effects of paclitaxel. Compound 1 is a substrate
of P-gp, as shown by the study where transiently transfected PC-3
cells, which lack this transporter, with a plasmid (pHaMDR-EGFP)
encoding P-gp fused to enhanced green fluorescent protein (EGFP).
Unlike HeLa cells, imaging of PC-3 cells treated with Compound 1 (1
.mu.M) revealed strong blue fluorescence in the absence of
verapamil (data not shown). However, in cells expressing green
fluorescent P-gp-EGFP, decreased blue fluorescence was observed,
dependent on the level of P-gp-EGFP expression, indicating that
Compound 1 is a potent substrate of this transporter. Treatment
with verapamil reversed this effect by blocking P-gp to prevent
efflux of Compound 1.
[0077] Studies were conducted to investigate the efflux of Compound
1 in HCT-15 cells that express high levels of P-gp. Compound 1 was
compared with rhodamine 123 (Rho123), a common P-gp substrate that
accumulates in mitochondria. As shown in FIGS. 6A-6D, cells were
analyzed after treatment with Compound 1 (1 .mu.M), Rho123 (1
.mu.M), and verapamil (0, 25, 100 .mu.M) by confocal microscopy and
flow cytometry. FIG. 6A shows an image from confocal laser scanning
and DIC microscopy of HCT-15 cells treated with the P-gp substrates
rhodamine 123 (1 .mu.M) with the vehicle only, left 2 panels, and
with verapamil (25 .mu.M, 1 h). FIG. 6B shows an image from
confocal laser scanning and DIC microscopy of HCT-15 cells treated
with the P-gp substrates Compound 1 (1 .mu.M) with the vehicle
only, left 2 panels, and with verapamil (25 .mu.M, 1 hr). FIG. 6C
shows data from an analysis of HCT-15 cells treated with rhodamine
123 (1 .mu.M) (left panels) or Compound 1 (1 .mu.M) (right panels)
by flow cytometry, were concentrations of added verapamil are
shown. FIG. 6D shows data from an analysis of HeLa cells treated
with rhodamine 123 (1 .mu.M) (left panels) or Compound 1 (1 .mu.M)
(right panels) by flow cytometry, where concentrations of added
verapamil are shown. Whereas the fluorescence of Rho123 increased
by only 3-fold in the presence of verapamil (at 25 or 100 .mu.M),
the fluorescence of Compound 1 increased by 7-fold at 25 .mu.M and
15-fold at 100 .mu.M verapamil. Moreover, in HeLa cells, Compound 1
could detect low levels of P-gp activity in HeLa cells that were
undetectable by Rho123 (FIG. 6D), enhancing fluorescence by 10-fold
at 25 .mu.M verapamil and 23-fold at 100 .mu.M verapamil,
indicating that Compound 1 can be used as a uniquely sensitive
sensor of this efflux transporter.
[0078] In contrast to Flutax-2.RTM., which is only weakly
cytotoxic, and exhibits relatively low cellular permeability due to
the high polarity of the appended OG fluorophore, the more
drug-like Compound 1 substantially recapitulates aspects of the
cytotoxic, tubulin-binding, and P-gp-mediated efflux activity of
the parent anticancer drug. Because PB can be efficiently detected
and analyzed by confocal microscopy and flow cytometry, this probe
offers a new tool for studies of the paradoxical anticancer effects
of paclitaxel. Thus, the taxane PB fluorophores are superior to
Flutax-2.RTM.. As such, Compounds 1-3 can be used to study the
proliferation rate paradox associated with the parent drug.
[0079] The uptake of Compound 1 (PB-Gly-Taxol, at 1 .mu.M, 1 h,
200,000 cells/200 .mu.L) by different cell lines was studied and
quantified by using flow cytometry. The study was done with
different concentrations of verapamil or no verapamil such as, 0
.mu.M, 25 .mu.M, 100 .mu.M, for the data bars going from left to
right for each cell type. As shown in FIG. 7, the inclusion of
verapamil increased the uptake. FIG. 7 shows a graph of data for
the uptake of Compound 1 into the HeLa, Ramos, Raji, HL-60, and
Jurkat cell lines, and the update was increased by adding
increasing amounts of verapamil. These experiments demonstrate the
high sensitivity of Compound 1 (PB-Gly-Taxol) for detection of P-gp
in a wide variety of cell lines including both adherent and
suspension cells.
[0080] Additional information regarding the synthesis and
characterization of Compounds 1-3, and relevant data thereof is
included in the provisional application that is incorporated herein
by specific reference in its entirety.
[0081] While the previously discussed reaction schemes and data
included the compounds under Formula 1 where the R group is the
oxygen anion or hydroxide (e.g. forming the phenoxide), now the
compounds that fall under Formula 2 with the R1 and R2 group are
described. FIG. 8A shows reaction Scheme 2 that is performed in
order to prepare alternative coumarin analogues of Pacific Blue
that can be used to prepare the compounds under Formula 2. It
should be recognized that the reaction in Scheme 2 may be modulated
so that the R group can vary to be any of the substituents recited
herein. Then, FIG. 8B shows the reaction Scheme 3 that is performed
to prepare the taxane (e.g., paclitaxel) into the taxane PB
fluorophores under Formula 2. Also, it should be recognized that
any taxane may be prepared as shown to be the reagent in order to
prepare a wide variety of taxane PB fluorophores.
[0082] An experiment was conducted to determine the toxicity of
paclitaxel (Taxol.RTM.), PB-Gly-Taxol (Compound 1),
PB-NHMe-Gly-Taxol (Compound 21), and PB-NH2-Gly-Taxol (Compound 22)
in HeLa cells. FIG. 9 shows a graph for the toxicity of paclitaxel
(Taxol.RTM.), PB-Gly-Taxol (Compound 1), PB-NHMe-Gly-Taxol
(Compound 21), and PB-NH2-Gly-Taxol (Compound 22), which shows the
cellular viability (percentage) versus concentration. The IC50s
were also determined. The data shows that PB-Gly-Taxol (Compound 1)
is less toxic than PB-NHMe-Gly-Taxol (Compound 21), but these
specific compounds are less toxic than paclitaxel. As such,
PB-NHMe-Gly-Taxol (Compound 21) and PB-NH2-Gly-Taxol (Compound 22)
may exhibit beneficial properties as anticancer agents compared
with PB-Gly-Taxol (Compound 1). Also, because PB-NHMe-Gly-Taxol
(Compound 21) and PB-NH2-Gly-Taxol (Compound 22) show a unique
pattern of subcellular accumulation in the endoplasmic reticulum
these compounds may be used as a probe for cytotoxic effects of
paclitaxel targets in the endoplasmic reticulum. Because these and
related compounds associate with internal membranes of the
endoplasmic reticulum, they may further exhibit beneficial
properties as therapeutic agents by avoiding efflux mediated by
P-gp, a transporter found at the cellular plasma membrane, and yet
still allow exchange between the endoplasmic reticulum and
microtubules found throughout cells to manifest toxicity.
[0083] Additionally, the absorbance spectra were determined for 30
.mu.M PB-NHMe-Gly-Taxol (Compound 21, RNHMe) and PB-NH2-Gly-Taxol
(Compound 22, RNH2) in PBS and 1-octanol. FIG. 10A shows the
absorbance spectra for PB-NHMe-Gly-Taxol (Compound 21, RNHMe) and
PB-NH2-Gly-Taxol (Compound 22, RNH2) in PBS and 1-octanol along
with the maximum absorbance wavelength. It should be recognized
that for Formula 1, Compound 21 has R being --NH--CH.sub.3 and
Compound 22 has R being --NH.sub.2.
[0084] Also, the emission spectra were determined for 50 nM
PB-NHMe-Gly-Taxol (Compound 21, RNHMe) and PB-NH.sub.2-Gly-Taxol
(Compound 22, RNH.sub.2) in PBS and 1-octanol. FIG. 10B shows the
emission spectra for PB-NHMe-Gly-Taxol (Compound 21, RNHMe) and
PB-NH2-Gly-Taxol (Compound 22, RNH.sub.2) in PBS and 1-octanol
along with the maximum emission wavelength.
[0085] Experiments were conducted to determine whether or not
PB-NHMe-Gly-Taxol (Compound 21) is a substrate of P-glycoprotein in
HeLa cells. Confocal microscopy revealed that PB-NHMe-Gly-Taxol and
PB-NH2-Gly-Taxol are not substrates of P-glycoprotein and
accumulate in the endoplasmic reticulum of live cells, as shown in
FIG. 11. The low fluorescence of these compounds in aqueous
solution compared to membrane mimics such as 1-octanol (FIG. 10)
may limit visualization of microtubules in cells with these
compounds. The observation that these compounds retain high
cytotoxicity (suggesting interaction with microtubules that cannot
be readily visualized) and avoid efflux by P-gp, while accumulating
in the endoplasmic reticulum, suggest that they may exhibit unique
and beneficial therapeutic effects compared with paclitaxel. FIG.
11 shows images of a P-glycoprotein response to PB-Gly-Taxol and
PB-NHMe-Gly-Taxol, with or without verapamil. The experiment was
conducted by 1 hour incubation with HeLa cells, followed by a wash
and then imaging using a 25% laser power with excitation at 405 nm
and emission at 425-500 nm. The top left Panel A shows 1 .mu.M
PB-Gly-Taxol (Compound 1), and Panel A1 is the same as Panel A plus
25 verapamil. Panel B shows 10 nM PB-NHMe-Gly-Taxol, and Panel B1
is the same as Panel B plus 25 .mu.M verapamil. Panel C shows 100
nM PB-NHMe-Gly-Taxol, and Panel C1 is the same as Panel C plus 25
.mu.M verapamil. Panel D shows 1 .mu.M PB-NHMe-Gly-Taxol, and Panel
D1 is the same as Panel D plus 25 .mu.M verapamil.
[0086] Experiments were conducted to determine whether or not
PB-NHMe-Gly-Taxol (Compound 21) can colocalize with Flutax-2.RTM.
on microtubules in live cells by confocal microscopy. Confocal
microscopy indicated that PB-NHMe-Gly-Taxol (Compound 21) does not
appear to colocalize with Flutax-2.RTM. on microtubules in live
HeLa cells but rather has a unique pattern of subcellular
localization that could benefit therapeutic applications by
enhancing the residence time in cancer cells. The colocalization
assay included 10 nM of PB-NHMe-Gly-Taxol (Compound 21) with 5
.mu.M Flutax-2.RTM. and 25 verapamil in HeLa cells for 1 hour. FIG.
12 shows different images in the panels for different excitation
wavelengths and emission wavelengths, as well as overlays and the
DIC microscopy images. FIG. 12 shows PB-NHMe-Gly-Taxol (Compound
21) does not colocalize with Flutax-2.RTM. on microtubules in live
HeLa cells.
[0087] Studies were conducted to determine if PB-NHMe-Gly-Taxol
(Compound 21) interacted with the endoplasmic reticulum, and was
compared to red fluorescent endoplasmic reticulum probe HRA (hexyl
resorufamine) in live cells using fluorescent microscopy. The
colocalization assay was conducted with 100 nM PB-NHMe-Gly-Taxol
with 1 .mu.M HRA in HeLa for 1 h. FIG. 13 shows different images
with different wavelengths and the overlay thereof as well as the
DIC image. This shows that PB-NHMe-Gly-Taxol colocalizes with the
red fluorescent endoplasmic reticulum probe HRA in live HeLa
cells.
[0088] Studies were conducted to determine whether or not
PB-NHMe-Gly-Taxol (Compound 21) is potent as an endoplasmic
reticulum probe. The studies compared the potency as an endoplasmic
reticulum probe with commercial ER probe ER Tracker Blue-White DPX.
The amounts of PB-NHMe-Gly-Taxol (Compound 21) and ER Tracker
Blue-White DPX were increased by Panels A through A3 showing
PB-NHMe-Gly-Taxol (Compound 21) and Panels B through B3 showing ER
Tracker Blue-White DPX. Panels A and B used 1 nM of the compounds,
Panels A1 and B1 used 10 nM of the compounds, Panels A2 and B2 used
100 nM of the compounds, and Panels A3 and B3 used 1 .mu.M of the
compounds. FIG. 14 shows confocal microscopy images of endothelium
reticulum interaction at increasing concentrations for
PB-NHMe-Gly-Taxol (Compound 21, A-A3) and ER Tracker Blue-White DPX
(B-B3). Accordingly, PB-NHMe-Gly-Taxol (Compound 21) is more potent
as an endothelium reticulum probe compared to ER Tracker Blue-White
DPX. These results demonstrate that modification of taxoids with
coumarin-derived fluorophores can provide a novel method to reduce
the sensitivity of taxoids to efflux by P-glycoprotein by causing
accumulation in the endoplasmic reticulum while retaining
substantial toxicity against cancer cells.
[0089] Additionally, studies were done that show that it is
surprising and unexpected that the compounds described herein, such
as Compound 2 (PB-.beta.-Ala-Taxol) can bind to microtubules better
than when paclitaxel is coupled to a different fluorophore, such as
when forming iPr-Rhodol-.beta.-Ala-Taxol. As such, small changes to
the fluorophore core structure can prevent the paclitaxel
fluorescent derivative from performing or acting the same as
paclitaxel, and such small changes can reduce binding of
fluorescent taxanes to microtubules. As such, Compound 2
(PB-.beta.-Ala-Taxol) was compared to iPr-Rhodol-.beta.-Ala-Taxol
with HeLa cells transfected with mCherry-tubulin. In contrast to
PB-.beta.-Ala-Taxol, the iPr-Rhodol-.beta.-Ala-Taxol does not bind
extensively to microtubules, but rather appears to accumulate
predominantly in the endoplasmic reticulum of living HeLa cells, in
this case without engendering substantial cytotoxicity, unlike the
more toxic coumarin-linked taxoids PB-NHMe-Taxol and
PB-NH.sub.2-Taxol that also accumulate in this organelle.
##STR00016##
[0090] Definitions:
[0091] The term "alkyl" or "aliphatic" as used herein refers to a
branched or unbranched saturated hydrocarbon group typically
although not necessarily containing 1 to about 24 carbon atoms,
such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl,
t-butyl, octyl, decyl, and the like, as well as cycloalkyl groups
such as cyclopentyl, cyclohexyl, and the like. Generally, although
again not necessarily, alkyl groups herein contain 1 to about 18
carbon atoms, or 1 to about 12 carbon atoms. The term "lower alkyl"
intends an alkyl group of 1 to 6 carbon atoms. Substituents
identified as "C.sub.1-C.sub.6 alkyl" or "lower alkyl" contains 1
to 3 carbon atoms, and such substituents contain 1 or 2 carbon
atoms (i.e., methyl and ethyl). "Substituted alkyl" refers to alkyl
substituted with one or more substituent groups, and the terms
"heteroatom-containing alkyl" and "heteroalkyl" refer to alkyl in
which at least one carbon atom is replaced with a heteroatom, as
described in further detail infra. If not otherwise indicated, the
terms "alkyl" and "lower alkyl" include linear, branched, cyclic,
unsubstituted, substituted, and/or heteroatom-containing alkyl or
lower alkyl, respectively.
[0092] The terms "alkenyl" as used herein refers to a linear,
branched or cyclic hydrocarbon group of 2 to about 24 carbon atoms
containing at least one double bond, such as ethenyl, n-propenyl,
isopropenyl, n-butenyl, isobutenyl, octenyl, decenyl, tetradecenyl,
hexadecenyl, eicosenyl, tetracosenyl, and the like. Generally,
although again not necessarily, alkenyl groups herein contain 2 to
about 18 carbon atoms, or 2 to 12 carbon atoms. The term "lower
alkenyl" intends an alkenyl group of 2 to 6 carbon atoms, and the
specific term "cycloalkenyl" intends a cyclic alkenyl group, or
having 5 to 8 carbon atoms. The term "substituted alkenyl" refers
to alkenyl substituted with one or more substituent groups, and the
terms "heteroatom-containing alkenyl" and "heteroalkenyl" refer to
alkenyl in which at least one carbon atom is replaced with a
heteroatom. If not otherwise indicated, the terms "alkenyl" and
"lower alkenyl" include linear, branched, cyclic, unsubstituted,
substituted, and/or heteroatom-containing alkenyl and lower
alkenyl, respectively.
[0093] The term "alkynyl" as used herein refers to a linear or
branched hydrocarbon group of 2 to 24 carbon atoms containing at
least one triple bond, such as ethynyl, n-propynyl, and the like.
Generally, although again not necessarily, alkynyl groups herein
contain 2 to about 18 carbon atoms, or 2 to 12 carbon atoms. The
term "lower alkynyl" intends an alkynyl group of 2 to 6 carbon
atoms. The term "substituted alkynyl" refers to alkynyl substituted
with one or more substituent groups, and the terms
"heteroatom-containing alkynyl" and "heteroalkynyl" refer to
alkynyl in which at least one carbon atom is replaced with a
heteroatom. If not otherwise indicated, the terms "alkynyl" and
"lower alkynyl" include linear, branched, unsubstituted,
substituted, and/or heteroatom-containing alkynyl and lower
alkynyl, respectively.
[0094] The term "alkoxy" as used herein intends an alkyl group
bound through a single, terminal ether linkage; that is, an
"alkoxy" group may be represented as --O-alkyl where alkyl is as
defined above. A "lower alkoxy" group intends an alkoxy group
containing 1 to 6 carbon atoms, and includes, for example, methoxy,
ethoxy, n-propoxy, isopropoxy, t-butyloxy, etc. Substituents
identified as "C.sub.1-C.sub.6 alkoxy" or "lower alkoxy" herein
contain 1 to 3 carbon atoms, and such substituents contain 1 or 2
carbon atoms (i.e., methoxy and ethoxy).
[0095] The term "aryl" as used herein, and unless otherwise
specified, refers to an aromatic substituent containing a single
aromatic ring or multiple aromatic rings that are fused together,
directly linked, or indirectly linked (such that the different
aromatic rings are bound to a common group such as a methylene or
ethylene moiety). Examples of aryl groups contain 5 to 20 carbon
atoms, and aryl groups contain 5 to 14 carbon atoms. Exemplary aryl
groups contain one aromatic ring or two fused or linked aromatic
rings, e.g., phenyl, naphthyl, biphenyl, diphenylether,
diphenylamine, benzophenone, and the like. "Substituted aryl"
refers to an aryl moiety substituted with one or more substituent
groups, and the terms "heteroatom-containing aryl" and "heteroaryl"
refer to aryl substituent, in which at least one carbon atom is
replaced with a heteroatom, as will be described in further detail
infra. If not otherwise indicated, the term "aryl" includes
unsubstituted, substituted, and/or heteroatom-containing aromatic
sub stituents.
[0096] The term "aryloxy" as used herein refers to an aryl group
bound through a single, terminal ether linkage, wherein "aryl" is
as defined above. An "aryloxy" group may be represented as --O-aryl
where aryl is as defined above. Examples of aryloxy groups contain
5 to 20 carbon atoms, and aryloxy groups contain 5 to 14 carbon
atoms. Examples of aryloxy groups include, without limitation,
phenoxy, o-halo-phenoxy, m-halo-phenoxy, p-halo-phenoxy,
o-methoxy-phenoxy, m-methoxy-phenoxy, p-methoxy-phenoxy,
2,4-dimethoxy-phenoxy, 3,4,5-trimethoxy-phenoxy, and the like.
[0097] The term "alkaryl" refers to an aryl group with an alkyl
substituent, and the term "aralkyl" refers to an alkyl group with
an aryl substituent, wherein "aryl" and "alkyl" are as defined
above. Examples of aralkyl groups contain 6 to 24 carbon atoms, and
aralkyl groups contain 6 to 16 carbon atoms. Examples of aralkyl
groups include, without limitation, benzyl, 2-phenyl-ethyl,
3-phenyl-propyl, 4-phenyl-butyl, 5-phenyl-pentyl, 4-phenyl
cyclohexyl, 4-benzylcyclohexyl, 4-phenylcyclohexylmethyl,
4-benzylcyclohexylmethyl, and the like. Alkaryl groups include, for
example, p-methylphenyl, 2,4-dimethylphenyl, p-cyclohexylphenyl,
2,7-dimethylnaphthyl, 7-cyclooctylnaphthyl,
3-ethyl-cyclopenta-1,4-diene, and the like.
[0098] The term "cyclic" refers to alicyclic or aromatic
substituents that may or may not be substituted and/or heteroatom
containing, and that may be monocyclic, bicyclic, or
polycyclic.
[0099] The terms "halo" and "halogen" are used in the conventional
sense to refer to a chloro, bromo, and fluoro or iodo
substituent.
[0100] The term "heteroatom-containing" as in a
"heteroatom-containing alkyl group" (also termed a "heteroalkyl"
group) or a "heteroatom-containing aryl group" (also termed a
"heteroaryl" group) refers to a molecule, linkage or substituent in
which one or more carbon atoms are replaced with an atom other than
carbon, e.g., nitrogen, oxygen, sulfur, phosphorus or silicon,
typically nitrogen, oxygen or sulfur. Similarly, the term
"heteroalkyl" refers to an alkyl substituent that is
heteroatom-containing, the term "heterocyclic" refers to a cyclic
substituent that is heteroatom-containing, the terms "heteroaryl"
and heteroaromatic" respectively refer to "aryl" and "aromatic"
substituents that are heteroatom-containing, and the like. Examples
of heteroalkyl groups include alkoxyaryl, alkylsulfanyl-substituted
alkyl, N-alkylated amino alkyl, and the like. Examples of
heteroaryl substituents include pyrrolyl, pyrrolidinyl, pyridinyl,
quinolinyl, indolyl, pyrimidinyl, imidazolyl, 1,2,4-triazolyl,
tetrazolyl, etc., and examples of heteroatom-containing alicyclic
groups are pyrrolidino, morpholino, piperazino, piperidino,
etc.
[0101] The term "hydrocarbyl" refers to univalent hydrocarbyl
radicals containing 1 to about 30 carbon atoms, or 1 to about 24
carbon atoms, or 1 to about 18 carbon atoms, or about 1 to 12
carbon atoms, including linear, branched, cyclic, saturated, and
unsaturated species, such as alkyl groups, alkenyl groups, aryl
groups, and the like. "Substituted hydrocarbyl" refers to
hydrocarbyl substituted with one or more substituent groups, and
the term "heteroatom-containing hydrocarbyl" refers to hydrocarbyl
in which at least one carbon atom is replaced with a heteroatom.
Unless otherwise indicated, the term "hydrocarbyl" is to be
interpreted as including substituted and/or heteroatom-containing
hydrocarbyl moieties.
[0102] By "substituted" as in "substituted alkyl," "substituted
aryl," and the like, as alluded to in some of the aforementioned
definitions, is meant that in the alkyl, aryl, or other moiety, at
least one hydrogen atom bound to a carbon (or other) atom is
replaced with one or more non-hydrogen substituents.
[0103] In addition, the aforementioned functional groups may, if a
particular group permits, be further substituted with one or more
additional functional groups or with one or more hydrocarbyl
moieties such as those specifically enumerated above. Analogously,
the above-mentioned hydrocarbyl moieties may be further substituted
with one or more functional groups or additional hydrocarbyl
moieties such as those specifically enumerated.
[0104] When the term "substituted" appears prior to a list of
possible substituted groups, it is intended that the term apply to
every member of that group. For example, the phrase "substituted
alkyl, alkenyl, and aryl" is to be interpreted as "substituted
alkyl, substituted alkenyl, and substituted aryl." Analogously,
when the term "heteroatom-containing" appears prior to a list of
possible heteroatom-containing groups, it is intended that the term
apply to every member of that group. For example, the phrase
"heteroatom-containing alkyl, alkenyl, and aryl" is to be
interpreted as "heteroatom-containing alkyl, heteroatom-containing
alkenyl, and heteroatom-containing aryl."
[0105] All other chemistry terms are defined as known in the
art.
[0106] One skilled in the art will appreciate that, for this and
other processes and methods disclosed herein, the functions
performed in the processes and methods may be implemented in
differing order. Furthermore, the outlined steps and operations are
only provided as examples, and some of the steps and operations may
be optional, combined into fewer steps and operations, or expanded
into additional steps and operations without detracting from the
essence of the disclosed embodiments.
[0107] The present disclosure is not to be limited in terms of the
particular embodiments described in this application, which are
intended as illustrations of various aspects. Many modifications
and variations can be made without departing from its spirit and
scope, as will be apparent to those skilled in the art.
Functionally equivalent methods and apparatuses within the scope of
the disclosure, in addition to those enumerated herein, will be
apparent to those skilled in the art from the foregoing
descriptions. Such modifications and variations are intended to
fall within the scope of the appended claims. The present
disclosure is to be limited only by the terms of the appended
claims, along with the full scope of equivalents to which such
claims are entitled. It is to be understood that this disclosure is
not limited to particular methods, reagents, compounds compositions
or biological systems, which can, of course, vary. It is also to be
understood that the terminology used herein is for the purpose of
describing particular embodiments only, and is not intended to be
limiting.
[0108] With respect to the use of substantially any plural and/or
singular terms herein, those having skill in the art can translate
from the plural to the singular and/or from the singular to the
plural as is appropriate to the context and/or application. The
various singular/plural permutations may be expressly set forth
herein for sake of clarity.
[0109] It will be understood by those within the art that, in
general, terms used herein, and especially in the appended claims
(e.g., bodies of the appended claims) are generally intended as
"open" terms (e.g., the term "including" should be interpreted as
"including but not limited to," the term "having" should be
interpreted as "having at least," the term "includes" should be
interpreted as "includes but is not limited to," etc.). It will be
further understood by those within the art that if a specific
number of an introduced claim recitation is intended, such an
intent will be explicitly recited in the claim, and in the absence
of such recitation no such intent is present. For example, as an
aid to understanding, the following appended claims may contain
usage of the introductory phrases "at least one" and "one or more"
to introduce claim recitations. However, the use of such phrases
should not be construed to imply that the introduction of a claim
recitation by the indefinite articles "a" or "an" limits any
particular claim containing such introduced claim recitation to
embodiments containing only one such recitation, even when the same
claim includes the introductory phrases "one or more" or "at least
one" and indefinite articles such as "a" or "an" (e.g., "a" and/or
"an" should be interpreted to mean "at least one" or "one or
more"); the same holds true for the use of definite articles used
to introduce claim recitations. In addition, even if a specific
number of an introduced claim recitation is explicitly recited,
those skilled in the art will recognize that such recitation should
be interpreted to mean at least the recited number (e.g., the bare
recitation of "two recitations," without other modifiers, means at
least two recitations, or two or more recitations). Furthermore, in
those instances where a convention analogous to "at least one of A,
B, and C, etc." is used, in general such a construction is intended
in the sense one having skill in the art would understand the
convention (e.g., "a system having at least one of A, B, and C"
would include but not be limited to systems that have A alone, B
alone, C alone, A and B together, A and C together, B and C
together, and/or A, B, and C together, etc.). In those instances
where a convention analogous to "at least one of A, B, or C, etc."
is used, in general such a construction is intended in the sense
one having skill in the art would understand the convention (e.g.,
"a system having at least one of A, B, or C" would include but not
be limited to systems that have A alone, B alone, C alone, A and B
together, A and C together, B and C together, and/or A, B, and C
together, etc.). It will be further understood by those within the
art that virtually any disjunctive word and/or phrase presenting
two or more alternative terms, whether in the description, claims,
or drawings, should be understood to contemplate the possibilities
of including one of the terms, either of the terms, or both terms.
For example, the phrase "A or B" will be understood to include the
possibilities of "A" or "B" or "A and B."
[0110] In addition, where features or aspects of the disclosure are
described in terms of Markush groups, those skilled in the art will
recognize that the disclosure is also thereby described in terms of
any individual member or subgroup of members of the Markush
group.
[0111] As will be understood by one skilled in the art, for any and
all purposes, such as in terms of providing a written description,
all ranges disclosed herein also encompass any and all possible
subranges and combinations of subranges thereof. Any listed range
can be easily recognized as sufficiently describing and enabling
the same range being broken down into at least equal halves,
thirds, quarters, fifths, tenths, etc. As a non-limiting example,
each range discussed herein can be readily broken down into a lower
third, middle third and upper third, etc. As will also be
understood by one skilled in the art all language such as "up to,"
"at least," and the like include the number recited and refer to
ranges which can be subsequently broken down into subranges as
discussed above.
[0112] Finally, as will be understood by one skilled in the art, a
range includes each individual member. Thus, for example, a group
having 1-3 cells refers to groups having 1, 2, or 3 cells.
Similarly, a group having 1-5 cells refers to groups having 1, 2,
3, 4, or 5 cells, and so forth.
[0113] From the foregoing, it will be appreciated that various
embodiments of the present disclosure have been described herein
for purposes of illustration, and that various modifications may be
made without departing from the scope and spirit of the present
disclosure. Accordingly, the various embodiments disclosed herein
are not intended to be limiting, with the true scope and spirit
being indicated by the following claims.
* * * * *